Acetate, which is not transported across the mitochondrial membrane, reacts with aspartic acid within the mitochondrion to form N-acetylaspartic acid (NAA). NAA is subsequently transported across the mitochondrial membrane to the cytosol where it is cleaved to acetate and aspartic acid and the acetate used as the sole carbon source for fatty acid and cholesterol synthesis. To measure NAA concentration in rat brain regions, the brain was removed, dissected into brain regions, and the tissue frozen at �70�C. To quantitate NAA, the tissue samples were homogenized, a known quantity of [15N]NAA added to the homogenate, NAA was partially purified and then quantitated by gas chromatography-mass spectroscopy. Between 10 and approximately 20 days, NAA levels increased rapidly in all brain regions regardless of whether tissue wet weight, tissue dry weight or soluble protein was used in determining NAA levels. At later ages, NAA levels increased at a slower rate if calculated by tissue wet weight. For these later ages, when either tissue dry weight or soluble protein to calculate NAA levels, the NAA levels were constant. During the period when NAA levels are increasing rapidly, myelination is also proceeding at a rapid pace. By comparing white and grey matter regions of the brain, our results suggest that NAA in oligodendrocytes contribute to brain NAA levels. Furthermore, decreased NAA levels, observed in certain brain disorders, should not be assumed, a priori, to be changes in neuronal NAA levels. (This work was supported in part by grant HD29926 from NICHD.)